Foreign stingers: South American freshwater river stingrays Potamotrygon spp. established in Indonesia

The pet trade is known to be one of the most important pathways of aquatic non-native species introduction and Indonesia is a significant trade partner. Popular ornamental South American river stingrays (Potamotrygon spp.) were introduced to Indonesia in the 1980s and the culture was established. Here we present a detailed Indonesian market and aquaculture survey, the volume of trade between January 2020 and June 2022, and the list of customer countries with the total amount of imported stingrays. Climate similarities between the native range of P. motoro and P. jabuti and Indonesia were analysed. A significant number of areas of Indonesian islands were identified as suitable for the establishment of this species. This was confirmed by the first record of probably established populations in the Brantas River (Java). In total 13 individuals including newborns were captured. The culture of potamotrygonid stingrays is unregulated in Indonesia, and the risk of the establishment of this predator and its potential spread is alarming for wildlife. Moreover, the first case of envenomation caused by Potamotrygon spp. in the wild outside of South America was recorded. The current condition is predicted as the ‘tip of the iceberg’ and continuous monitoring and mitigation of risks are strongly recommended.

www.nature.com/scientificreports/ be expected as potentially well-established in Java, Bali, and Nusa Tenggara, and some in Sumatra, Kalimantan, Sulawesi, Maluku Islands and Papua.
Records in the wild. In total, 13 individuals of Potamotrygon spp. (three morphologically identified as P. motoro, six as P. jabuti) were captured by local fishermen using ' Ayap' (traditional fishing gear which is similar to big bottom barless dipnet but made from bamboo; the mesh size ranged from 2 to 8 cm; the fisherman use the Ayap from riverbank when the water level is high or during flood, holding it in the water and waiting for fish drifting into the Ayap; when fish where stuck in the net, fisherman lift the Ayap and the fish are caught), angling, castnet, and gillnet in the Brantas River, East Java Province (Fig. 2c, 3, Table 1). Also, newborns and a gravid female of P. jabuti were recorded and thus, reproduction is occurring. The furthest distance between findings was more than 40 km. The voucher specimen was identified as Potamotrygon motoro.
The voucher specimen (P. motoro, adult male, total body length 370 mm, disc width 230 mm) was caught on 28 November 2021 using Ayap and kept in an aquarium by a local angler (Fig. 4a). In concordance with previously published morphological analysis 44 , disc was sub-circular with small tips on the anterior margin. Eyes bulging dorsally and relatively large (4% DW). Dorsally covered with tricolour ocelli (Fig. 4d), evenly distributed. The central region of the dorsal disc presents large irregular shapes due to unidentified derm disease (Fig. 4a,b). Pelvic fins are present over the posterior margin of the disc. The tail is relatively short, thick, and punctured on the posterior margin of the sting. One row of curved tail spines with a large base (Fig. 4c). Dermal denticles are present on the dorsal region of the disc and tail, various sizes covered on the disc, but larger denticles present on the central region of the disc, star-shaped denticles with dichotomous ridges present in large denticles, small denticles often with monochotomous ridges or 'x' mark-shaped denticles. The ventral disc is whitish with a black and grey irregular shape across the central region, outer margin covered with dark-grey colouration. This voucher specimen is deposited in Museum Zoologicum Bogoriense (MZB), Java, Indonesia, under the coll. No. MZB.26608.
Type habitats of P. motoro and P. jabuti in the Brantas River system consist of three areas as the main stream (Brantas River), floodplain, and irrigation canals connected to Brantas River. Water discharges of the main river correlated with dam activity in the upper stream. The connectivity of the floodplain and the main river will be greatly increasing if the water level of the main river was increased. The water discharge in irrigation canals fluctuated following the water level on the reservoir at Njegu Dam and Serut Dam.
We have conducted a rapid qualitative assessment of the habitats in the Brantas River. The Brantas River considers resembling native habitats based on qualitative descriptions of climate (tropic), substrates, and type habitats. The presence of a floodplain and connectivity between the main river and floodplain provides diverse habitat ranges. Muddy and sandy substrates provide similar niches for foraging and burying activities 45 . The habitats in the main river consisted of lacustrine (reservoir) and riverine region (river). Individuals in the lacustrine region (upper stream of Njegu Dam and Serut Dam) were found in areas with mud substrate sporadically covered with leaf litter. Records in the riverine region were found in boulder, pebble, sand, and mud substrates. The voucher specimen was collected on the shore of the riverine region with sand and mud substrates (depth of approximately 0.5-1.5 m). The depth of the main river varies and the observed water clarity range between 0 and 15 cm (wet season). In several sections of the main river, there were observed submerged deposits of sand and pebbles.
The floodplain habitats are located near the main river in the Brantas River system. The gravid female of P. jabuti with four neonate individuals and one adult male were caught in floodplain habitats. Water depth tends to be shallower (0.5-1.0 m) than the main river and mud dominate the type of substrates. Submerged aquatic plants dominate the edges of the floodplain.
Irrigation canals are connected to the reservoir in the upper stream of the Brantas River. Water clarity tends to fluctuate following reservoir activity (water level, drifted substrates, flood, and dam activity). The majority of  www.nature.com/scientificreports/    Align with a precautionary approach, we considered this type of habitat as potentially invaded.  www.nature.com/scientificreports/ Brantas River basin is a national strategic river basin playing a vital role in driving Indonesia's economy. The Brantas River is the second-longest river (320 km) on the island of Java and crosses the province of East Java. The upper stream of the Brantas River originates from several mountains and springs around the highlands in East Java. The Brantas River flows through various plantation lands, and paddy fields, and is one of the sources of raw material for drinking water in most areas in East Java. Connectivity between main rivers and irrigation canals is located upstream, middle, and downstream. Potamotrygon spp. were recorded in the middle (Blitar Regency and Tulungagung Regency) of the Brantas River.
The diets of non-native Potamotrygon spp. in the Brantas River are only known from an interview from local anglers. Several anglers use worm bait (Lumbricus spp.) and shrimps (Macrobrachium spp.) for fishing the Potamotrygon spp. The voucher individual was kept in indoor aquarium for several days and had been fed by shrimps and fish (meat-pieces of Barbonymus spp. and Oreochromis spp.).
Anthropogenic activities in the surveyed area were observed to be diverse and intense. The activities of fishermen, recreational fishing and sand mining were observed in the central part of the Brantas River. These activities are associated with the direct use of water bodies and overlap with the localities of Potamotrygon spp. records. Sand mining activities have been carried out in the main river with shallow sand substrates, where Potamotrygon spp. were commonly found (for example, locality from confirmed individuals on 22 November 2021 and 31 December 2021 are approximately 50 m from the sand mining site). During the survey of this locality, one fisherman who was stung by a captured P. jabuti was found. The fisherman was hospitalized for two days after being stung two times (in the left and right forearms). The fisherman described the pain as 'worse than being stabbed by a dagger' .
International trade. Of all P. motoro traded globally, Indonesia imported 2,717 individuals (5.4%), while 122 of them (2.0%) were exported (based on Importer/Exporter-reported quantity of CITES data). Indonesia does not report any trade in P. motoro to CITES, hence the need to shift focus from exporter to importer- Data from the Indonesian trade analysis based on data from the Fish Quarantine and Inspection Agency, Ministry of Marine Affairs and Fisheries Republic of Indonesia present a different scenario. In total, 3903 individuals of P. motoro were imported within the period of January 2020 to June 2022 from Thailand, Taiwan, China, Malaysia, India, and Colombia (the majority of 3525 individuals were from Thailand) and 27,777 individuals were exported to 25 countries (with 7660 individuals delivered to Japan; Figs. 5, 6). Furthermore, 1001 individuals were traded domestically. The first P. motoro were imported to Indonesia via trade routes from South America by fish entrepreneurs in the 1980s. The first exported individual was delivered to Qatar in March 2021, the largest export was recorded in March 2022 when 4,545 individuals were shipped to 12 countries (Table 2).

Discussion
In Indonesia, an established, self-sustaining population of non-native Potamotrygon spp. was found in the Brantas River, Java. This same river also recorded a South American giant arapaima 21 . Obviously, this is what is called a Biodiversity Conservation Paradox 46 because both mentioned taxa are endangered in their native ranges, but are becoming invasive.
Indonesia has broad ranges of freshwaters from peat water to blackwater (similar to Negro River in low acidity and a huge river system 47 ) in Sumatra and Kalimantan (Indonesian part of Borneo) to white-transparent water in three zoogeography areas 48 . The rivers in Borneo and Sumatra are also habitats of native freshwater stingrays (Fluvitrygon spp. and Urogymnus polylepis) 49,50 . The occurrences of freshwater stingrays (despite different genera and families, potentially similar in their physiology) indicate the potential suitability of natural habitats for to be invaded in the future.
Besides escapes from home aquaria and farms, "Fang Sheng" rite, and pest control 17,21 , a new pathway of non-native species introduction is worth mentioning: affluent people intentionally release expensive animals to demonstrate their wealth and power. There is no clear evidence about the Potamotrygon spp. introduction pathway in the Brantas River. Humans value non-natives for a wide range of reasons-aesthetic, culinary, or diverse cultural reasons, among them, financial/social status 51 . With globalization, the trend is that the number of rich Indonesians will increase in the future and thus, maintain the cultural tradition of releasing expensive non-native animals.
Even if climate matching analysis is a preliminary estimation, it showed that the vast majority of Indonesian territory is suitable for further establishment of P. motoro, P. jabuti, and other potamotrygonin species. Since aquatic ornamental creatures are known to be transported across the Indonesian territory in huge quantities 40 , it is not surprising that P. motoro and P. jabuti are becoming established. The Brantas River resembles native stingray habitats based on qualitative descriptions of climate (tropic), substrates, habitat types, and food resources. Muddy and sandy substrates provide similar niches for foraging and burying activities 45 . The presence of a floodplain and connectivity between the main river and floodplain provides diverse habitat ranges. The record of gravid female at the floodplain confirmed that Potamotrygon spp. can use this habitat as nursery ground in the Brantas River. The distribution of Potamotrygon spp. was recorded across the floodplain and main river. This stingray is able to overcome artificial barriers (found in three locations separated by artificial barriers such as Upperstream Njegu Dam, Downstream Njegu Dam-Upperstream Serut Dam, and Downstream Serut Dam). It is likely that stingrays crossed the barriers during flood events. A similar situation occurred with introduced P. motoro and P. falkneri in Jupiá Dam, Upper Paraná River basin, Southeastern Brazil, attesting to the ability of stingrays to overcome artificial barriers such as hydro power plants 52 .
Potamotrygon spp. are a nocturnal predatory fish 53 . The interview-based evidence of broad range of food preferences of recorded non-native Potamotrygon spp. have slight similarity with the stomach content of P. motoro in Negro River 54 . It is known that younger individuals inhabit mostly sandy bottoms of depth no more than four meters while bigger ones migrate between deeper waters during the day and shallow areas at night 45 but we have no exact data confirming or denying this behaviour also in Indonesia. Because the pigmented and well-camouflaged wild phenotype only is traded in Indonesia and because of burrowing activity, detection of Potamotrygon spp. by fishermen and collectors could be difficult. Simply, this species, for having a cryptic colouring, can be easily neglected by locals at least until its abundance will be high. On the other hand, once detected, it can be targeted according to its high price.
There are no native species in Indonesia which can be able to mate with this non-native stingray. On the other hand, negative impacts on native biota and also the local community can be expected. They are regarded by native people in the Amazonia as venomous fishes 55 and responsible for frequent stings and the envenomation has also occasionally been reported by aquarium traders and owners [56][57][58] . Especially venomous species can threaten naïve animals and can easily hurt fishermen and all laypersons who will handle these stingrays which is supported by the record of envenomation. For instance, stingray accidents are considered a public health problem in Brazil 59 . Most of the injuries caused by fish in the country involved freshwater stingrays, especially from the Potamotrygonidae family, and the most affected people are fishermen, who are handling these animals daily, and bathers (children and adults), especially during the dry season in Amazonia, Paraguay and Lower Paraná river basins (i.e., native range) 60,61 . Moreover, accident data in these regions is underestimated because most fishermen do not report or even go to the hospital; some fishermen get used to be frequently sting. In the non-native range (i.e., Upper Paraná River basin), stingray envenomation appeared over the past 20 years. An important natural barrier-the Seven Falls of Guaíra (Paraná State, Southern Brazil)-served as a natural barrier www.nature.com/scientificreports/ preventing species of the Lower Paraná River to colonize regions of the Upper Paraná River. However, with the creation of the Itaipu hydroelectric dam in 1982, these falls were submerged, allowing the movement of several fish species upstream, including freshwater stingrays that are taking advantage of locks installed at the dam to expand their distribution area 62 . In the Upper course of the Paraná River, injuries are reported by inhabitants and also tourists in municipalities of Mato Grosso do Sul, Paraná and São Paulo states, who are often unaware of the presence of these non-native animals in the area 63 . As is our knowledge, this is the first case of envenomation caused by Potamotrygon spp. in the wild outside of their native and non-native locations in South America. Such a situation is alarming because Potamotrygon spp. can reach populated areas, where most inhabitants are unaware of how to prevent accidents and to treat wounds they may cause. If we consider the existence of numerous properties by hotels and the intense practice of fishing/tourism activity in the region of Brantas River and throughout the country, it is expected that the negative interaction between humans and Potamotrygon spp. will be more intense, causing important changes in epidemiological profiles of envenomation in Indonesia. Further potential encounters between Potamotrygon spp. and humans are highly probable and imminent also due to sand mining and fishing in the reported localities in the Brantas River. Moreover, harmful consequences can be expected due to the predatory feeding behaviour of Potamotrygon spp. Diet analysis showed that aquatic invertebrates such as molluscs (both gastropods and bivalves), crustaceans (mainly shrimps from family Palaemonidae), insects (mainly Ephemeroptera and Diptera) and vertebrates such as fish are consumed 64,65 . Also, the foraging behaviour of Potamotrygon spp. can impact the native fauna. Several species of rays forage disturb the substrate, also known as bioturbation 66,67 , with the use of a tactic we termed "undulate the disc and stir substrate" to uncover insect larvae, crabs, snails and small fish 68 . This activity stirs the substrate particles and discrete sediment clouds are formed near the foraging ray 69 . These clouds can catch the attention of nearby rare Akysis variegatus, endangered Rasbora lateristriata, protected Notopterus notopterus and tadpoles of the vulnerables Microhyla orientalis and Gonocephalus kuhlii that approach the ray to feed and thus, it can prey on them 70,71 . Freshwater stingrays can also change the diet preferences according to the prey availability in different hydrological seasons. Indeed, this easy adjustment for the available resources can be the main advantage in competition for food with native aquatic organisms. Furthermore, competition for utilizing food and space with native benthic organisms (such as Hemibagrus sp. and Mastacembelus sp.) is predicted if the populations of Potamotrygon spp. explode. Thus, it is obvious that this invader can negatively affect rich Indonesian aquatic biota in general and native, rare, protected, endemic, endangered, and vulnerable species in particular. In line with no predators of potamotrygonin species in their native range 66 , it is also a main problem for these invasive organisms in Indonesia because the control of their populations by native predators is really difficult.
Several parasites such as Digenea, Nematoda and Cestoda are known in Potamotrygon spp. 72,73 . Also, branchiuran fish lice Argulus juparanaensis was found to infest P. motoro in its native range [74][75][76] . Therefore, the parasitic infection of native/endemic species cannot be excluded in the case of Potamotrygon spp. spread in Indonesia.
The popularity of various species of Potamotrygon as an ornamental fish in Indonesia is obvious because it is produced in significant quantities. In the early part of this species' history in Indonesia, some individuals were imported from Malaysia and India. The current list of customer countries is wide with Japan, Malaysia and Germany as leaders. No individuals were delivered to the Czech Republic even if this country is perceived as one of the leading producers and traders of ornamental fish globally 37,77,78 . This is probably caused by the significant production of Potamotrygon spp. by domestic breeders in the Czech Republic (Dařbujan, H., 2022 pers. comm.). It is obvious that the formal evidence of traded P. motoro in Indonesia is undervalued by CITES and the real volume of the trade is much higher. This discrepancy in trade evidence increases the potential for P. motoro and similar species to be invasive in Indonesia.
What is alarming, the current Indonesian legislative framework is ineffective in preventing the future introductions of this freshwater stingray to new localities and islands across the Indonesian territory 14 . Thus, without significant improvement in the management and regulations, the spread of Potamotrygon spp. in Indonesia can be expected to be unlimited for ongoing years. As Reynolds et al. 22 mentioned, detailed surveys of the trade with potamotrygonids are required for future proposals for CITES listing and we believe that the presented findings can be helpful in this regard.
Presented records align with the habitat use of native potamotrygonids at the Paraná River Basin 45 . Generally, potamotrygonids can adapt to broad ranges of physiochemical parameters 79 and we assume that this ability is crucial in case of invasion in the Brantas River. Further spread of these species is thus expected and the locality has to be monitored continuously and complemented by physical-chemical parameters measurements. The current condition is predicted as the 'tip of the iceberg' invasion event. Thus, continuous monitoring is recommended for this species. Therefore, we emphasize the importance of training health professionals near risk areas, as well as reporting accidents through a kind of "Information System for Notifiable Diseases" as it is carried out in Brazil 61 . According to Brazilian experience, it is recommended to remove fragments, wound cleaning, and immersion of the injured limb in hot water for pain relief (approximately 60 °C-due to the heat-labile properties of some of the toxins in the venom and the vasodilation caused by the hot water helps to counter the intense vasoconstriction and resulting ischemia induced by the venom), antibiotics to prevent bacterial septicemia, gangrene, tetanus, local anaesthetic, and systemic analgesics. Prevention of introductions is the first and most cost-effective management option for the Indonesian aquarium trade. Intensive awareness of importers, wholesalers, retailers, aquarium hobbyists and the general public about why releasing non-native freshwater stingrays is risky for native biota, and why releasing in wild could become a public health problem is crucial. Pamphlets combined with lectures, as well as the dissemination of information through the media, social networks like Facebook, Twitter, Instagram, and WhatsApp and warning signs in places of intense tourism/agglomeration of people showing that it is necessary to drag the feet or use a stick or paddle to grope the substrate in order to blows away any freshwater rays are measures that can be effective in preventing the number of accidents by rays in different parts of Indonesia. www.nature.com/scientificreports/ Last but not least is to focus on other species of the genus Potamotrygon which are offered for sale as ornamental creatures in Indonesia and evaluate their invasion potential and their ability to cause harm to people's health. Additional effort should be placed on the other tropical countries that produce Potamotrygon species.

Methods
Data collection was carried out after the first author received non-formal information from social media regarding the presence of stingrays in the Brantas River, Java (Fig. 2d). An in-depth online desk study was conducted following 21 in several mass-media and social media outlets (Facebook ® , www. faceb ook. com; Instagram ® , www. insta gram. com). A survey including direct observations was carried out in December 2021 by inventorying stingray findings in the Brantas River (Blitar and Tulungagung Regency). Interviews with locals were conducted using the snowball method to qualitatively identify the impact of the invasion on human activities around the Brantas River. In total, 75 people (local non-fishermen who live near the river/locality: such as farmers, 'tambangan' people, i.e. person who utilized/organized traditional boat-bridge, local shop keepers; local fishermen: anglers, fishermen) were asked about the occurrence/locality of Potamotrygon/freshwater stingray, and only less than 10 providing the specific information about freshwater stingray. Informed consent was obtained from all interviewed people. If possible, individuals observed during the survey were documented and preserved using 96% alcohol. Authors complied with the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations. All methods, experiments, protocols, and survey were approved by the ethics committee of Centre for Coastal and Marine Resources Studies at the Bogor Agricultural University (IPB; No. 17/IT3/PL/2012).
Trade information was based on an online survey obtained from two e-commerce platforms (Tokopedia ® , www. tokop edia. com; Shopee ® , www. shopee. co. id). Keywords in Bahasa Indonesia and English (pari, pari air tawar, motoro, pari motoro, pari hias, pari black diamond, stingray, stingray motoro, pari marble) were generated and applied to search engines in each platform. The result from each keyword was screened based on the quality of photographs, stock availability, reliability and track record of the stores. The filtered result was synthesized into data and qualitatively described.
The MaxEnt model (v. 3.4.4, https:// biodi versi tyinf ormat ics. amnh. org/ open_ source/ maxent/), a maximum entropy model that is ideally suited to mapping species distributions is commonly used to predict alien species dispersion 80 since it represents a continuous probability surface of habitat suitability in the target region 19,21,81,82 . This model consists of bioclimatic variables that are characteristics derived from monthly temperatures and rainfall data that reflect yearly patterns, seasonality, and extremes that are crucial for species survival. Bioclimatic factors from the WorldClim database were used to simulate species distribution (v.2.0; https:// www. world clim. org) 83 with a spatial resolution of 30 s (∼ 1 km 2 ). The distribution pattern of aquatic species and environmental factors, particularly temperature, have been discovered to be related 9,84 . These environmental layers were assembled in QGIS 3.24 'Tisler' and released on 13 May 2022 (https:// qgis. org/ en/ site/) to ASCII format for use with the MaxEnt algorithm 85 . We calculated nine bioclimatic variables for both P. motoro and P. jabuti (Table 3), these represented the average, extreme and variation of temperature and precipitation and were widely used in ecological niche modelling. MaxEnt was trained using all nine bioclimatic variables with default features and regularization multipliers (Default model), which was based on empirical tuning studies 80 . As the cumulative output, a continuous map was generated and visualized in QGIS 3.24 'Tisler' . The MaxEnt model output a threshold value for P. motoro = 12.74 and P. jabuti = 16.32. If the value of the climate match reached or exceeded this threshold, this was interpreted as no evidence of climatic constraints to the survival of the species and was shown in red on the map. The value for the area under the receiver operator curve (AUC) for P. motoro was 0.981 and for P. jabuti, was 0.982 which means there was a 98% probability for both species that a random selection from presence records had a model score greater than a random selection from the absence records 82 .
The trade analysis includes a detailed survey of evidence of the domestic market in Indonesia and main customer regions such as Europe with the Czech Republic as a hub 37,77 . Data from the Czech Environmental Inspectorate, (www. cizp. cz), CITES (https:// trade. cites. org/) and Fish Quarantine and Inspection Agency (FQIA), Ministry of Marine Affairs and Fisheries Republic of Indonesia were obtained for this purpose and further interpreted. www.nature.com/scientificreports/ The species identification of captured individuals was based on morphological analysis and comparison with the latest morphological study 32,86 . Morphological characteristics were measured using a calliper (accuracy 0.1 mm) and microscope images (Dino-lite AM2111). All captured individuals were exploited by the local community as ornamentals or for human consumption with the exception of a voucher specimen.

Data availability
The datasets used and analysed during the current study available from the corresponding author on reasonable request.